Method of screening candidate compounds for susceptibility to biliary excretion

ABSTRACT

A method of screening a candidate compound for susceptibility to biliary excretion. The method includes the steps of providing a culture of hepatocytes, the culture having at least one bile canaliculus; exposing a candidate compound to the culture; and determining an amount of candidate compound in the at least one bile canaliculus, the amount of candidate compound in the at least one bile canaliculus indicating the susceptibility of the candidate compound to biliary excretion. Optionally, the culture of hepatocytes is a long-term culture in a sandwich configuration. The method is particularly applicable to the screening of multiple candidate compounds in a single effort.

RELATED APPLICATION INFORMATION

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 60/124,810, filed Mar. 17, 1999, the disclosureof which is incorporated herein by reference in its entirety.

GRANT STATEMENT

This invention was made in part from government support under Grant No.GM41935 from the National Institute of Health. Thus, the U.S. Governmenthas certain rights in the invention.

TECHNICAL FIELD

The present invention relates to a method of screening compounds whichare candidates primarily for use as therapeutic agents forsusceptibility to biliary excretion. More particularly, the presentinvention relates to an in vitro method of screening candidate compoundsfor susceptibility to biliary excretion. Compounds can be chosen for useas therapeutic agents for administration to humans and otherwarm-blooded vertebrates.

Table of Abbreviations AUC area under the curve BSEP bile salt exportpump Cl_(B) biliary clearance Cl_(in) intrinsic clearance cMOATcanalicular multispecific organic anion transporter CFDAcarboxyfluorescein diacetate DMEM Dulbecco's modified Eagle's mediumEDTA ethylenediamine tetraacetate HP Hewlett Packard HPLC highperformance liquid chromatography hr hour i.v. intravenous i.p.intraperitoneal K_(m) Michaelus-Menten constant for enzyme-substratereaction LC/MS liquid chromatography/mass spectrometry mg pr. milligramsprotein min minute MDR2 multidrug resistance protein 2 MRP2 multidrugresistance associated protein 2 Ntcp Na⁺/taurocholate cotransportingpolypeptide OATP1 organic ion anion transporting polypeptide 1 OATP2organic ion anion transporting polypeptide 2 P-gp P-glycoprotein SDstandard deviation UV ultraviolet UV/VIS ultraviolet/visible V_(max)maximum velocity of enzyme-catalyzed reaction

BACKGROUND ART

First-pass metabolism pertains to the absorption of therapeutic agents,drugs or other compounds into the portal blood supply that leads to theliver. When a drug is swallowed, the stomach and small intestine absorbit, with subsequent flow in the blood to the portal vein entry to theliver. The liver may then in turn rapidly absorb and metabolize the drugat high concentrations through the liver blood supply. Thus, largeamounts of the drug may never be seen by the systemic circulation ordrug effect site. Additionally, rapid metabolism via the first-passmetabolism route can lead to the formation of high plasma concentrationsof unwanted metabolites.

Thus, in the liver, therapeutic compositions are often undesirablyremoved from an animal's circulatory system in that they are taken up byhepatocytes (liver cells) and excreted in bile via the bile canaliculi.Uptake into the hepatocytes is mediated by transport systems endogenousto hepatocytes, including Ntcp and cMOAT. Such transporters movexenobiotics like therapeutic compositions as well as endogenouscompounds across the sinusoidal membrane of the hepatocytes. Bilecanaliculi are structures within liver tissue that receive excretedcomponents from the hepatocytes and transport the bile to a common bileduct for removal from the animal. Biliary excretion of substrates isthus a complex process involving translocation across the sinusoidalmembrane, movement through the cytoplasm, and transport across thecanalicular membrane.

The advent of combinatorial chemistry techniques has enabled theidentification of extremely high numbers of compounds that havepotential as therapeutic agents. However, assays for susceptibility tobiliary excretion that can rapidly identify those candidate compoundsthat have a lower potential for uptake by hepatocytes and excretionthrough bile canaliculi have lagged behind the pace of synthesis andscreening of pharmacological activities. Numerous in vivo (e.g. bileduct cannulated animals) and in vitro preparations (e.g. isolatedperfused livers, isolated hepatocytes, hepatocyte couplets, liver plasmamembrane vesicles and expressed transport proteins) have been used toinvestigate biliary excretion processes. See e.g. Oude Elferink et al.,Biochim. Biophys. Acta 1241:215-268, 1995.

Additionally, short-term (3-8 hour) cultured hepatocyte couplets havebeen employed to examine directly the biliary excretion of fluorescentcompounds utilizing fluorescence microscopy, as described by Graf andBoyer, J. Hepatol. 10:387-394,1990. However, the application of culturedhepatocyte couplets to study biliary excretion of xenobiotics is limitedin that the substrate must contain a fluorescent chromophore.

Long-term (typically more than 24 hour) cultured hepatocytes have beenreported to restore polarity with canalicular-like structures. See e.g.,Barth and Schwarz, Proc. Natl. Acad. Sci. 79:4985-4987, 1982; Maurice etal., J. Cell Sci. 90:79-92, 1988; Talamini et al., Hepatology25:167-172, 1997. Although primary hepatocytes maintained underconventional culture conditions have been used to study drug metabolismand hepatotoxicity, long-term cultures of hepatocytes have not been asuitable model for studying hepatobiliary transport. Particularly, asdescribed by Groothuis and Meijer, J. Heptaology 24(Suppl. 1):3-28, 1996and LeCluyse et al., Adv. Drug Del. Rev. 22:133-186, 1996, rapid loss ofliver-specific function, including hepatic transport properties, andfailure to establish normal bile canalicular networks and to maintainnormal hepatocyte morphology have been observed in such cultures.

Existing methods have not been demonstrated to be widely applicable toinvestigate human biliary excretion. In addition, existing approachescannot be used to examine efficiently biliary excretion processes for alarge number of drug candidates. Thus, there is a long-felt need for anassay to assess susceptibility of candidate compounds for hepatic uptakeand biliary excretion. Such an assay would facilitate elimination ofthose compounds with an undesirably high susceptibility for biliaryexcretion from further evaluation as therapeutic agents early in theevaluation process. Correspondingly, there is a long-felt need for therapid identification of suitable candidate compounds (i.e., compoundsthat are not susceptible to biliary excretion) for further testing astherapeutic agents.

SUMMARY OF THE INVENTION

A method of screening a candidate compound for susceptibility to biliaryexcretion is disclosed herein. The method comprises the steps ofproviding a culture of hepatocytes, the culture comprising at least onebile canaliculus having a canalicular space; exposing a candidatecompound to the culture; and determining an amount of the candidatecompound in the canalicular space of the at least one bile canaliculus,the amount of the candidate compound in the canalicular space of the atleast one bile canaliculus indicating the susceptibility of thecandidate compound to biliary excretion. The culture of hepatocytespreferably comprises a long-term culture in a sandwich configuration.

Accordingly, it is an object of the present invention to provide a rapidand inexpensive method of screening of candidate compounds forsusceptibility to biliary excretion.

It is a further object of the present invention to provide an in vitromethod of screening candidate compounds for susceptibility to biliaryexcretion.

It is yet a further object of the present invention to provide a methodof screening candidate compounds for susceptibility to biliary excretionwhich facilitates the screening of many candidate compounds in a singleeffort.

It is still a further object of the present invention to provide a highthroughput method of screening of candidate compounds for susceptibilityto biliary excretion.

Some of the objects of the invention having been stated herein above,other objects will become evident as the description proceeds, whentaken in connection with the accompanying Laboratory Examples andDrawings as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph depicting cumulative uptake of [³H]inulin (1 μM) instandard buffer (closed symbols) and Ca⁺⁺-free buffer (open symbols) inhepatocyte monolayers cultured for 3 hr;

FIG. 1B is a graph depicting cumulative uptake of [³H]inulin (1 μM) instandard buffer (closed symbols) and Ca⁺⁺-free buffer (open symbols) inhepatocytes cultured in a sandwich configuration for 96 hr;

FIG. 2A is a graph depicting cumulative uptake of [¹⁴C]salicylate (1 μM)in standard buffer (closed symbols) and Ca⁺⁺-free buffer (open symbols)in hepatocyte monolayers cultured for 3 hr;

FIG. 2B is a graph depicting cumulative uptake of [¹⁴C]salicylate (1 μM)in standard buffer (closed symbols) and Ca⁺⁺-free buffer (open symbols)in hepatocytes cultured in a sandwich configuration for 96 hr;

FIG. 3A is a graph depicting cumulative uptake of [³H]methotrexate (1μM) in standard buffer (closed symbols) and Ca⁺⁺-free buffer (opensymbols) in hepatocyte monolayers cultured for 3 hr;

FIG. 3B is a graph depicting cumulative uptake of [³H]methotrexate (1μM) in standard buffer (closed symbols) and Ca⁺⁺-free buffer (opensymbols) in hepatocytes cultured in a sandwich configuration for 96 hr;

FIG. 4A is a graph depicting cumulative uptake of[³H][D-pen^(2,5)]enkephalin (15 μM) in standard buffer (closed symbols)and Ca⁺⁺-free buffer (open symbols) in hepatocyte monolayers culturedfor 3 hr;

FIG. 4B is graph depicting cumulative uptake of[³H][D-pen^(2,5)]enkephalin (15 μM) in standard buffer (closed symbols)and Ca⁺⁺-free buffer (open symbols) in hepatocytes cultured in asandwich configuration for 96 hr;

FIG. 5A is a graph depicting cumulative uptake of [³H]tasurocholate (1μM) in standard buffer (closed symbols) and Ca⁺⁺-free buffer (opensymbols) in hepatocyte monolayers cultured for 3 hr;

FIG. 5B is a graph depicting cumulative uptake of [³H]taurocholate (1μM) in standard buffer (closed symbols) and Ca⁺⁺-free buffer (opensymbols) in hepatocytes cultured in a sandwich configuration for 96 hr;

FIG. 6A is a graph depicting the relationship between the percentage ofthe dose excreted in rat bile in vivo and the Biliary Excretion Index in96-hr sandwich cultured hepatocytes for the following model substrates:inulin (□), salicylate (♦), methotrexate (◯), [D-pen^(2,5)]enkephalin(▴), and taurocholate (). The Biliary Excretion Index was calculatedfrom the 10-min cumulative uptake data (FIGS. 1A-5B) based on Equation3. The broken line is the fit of a linear regression equation to thedata;

FIG. 6B is a graph depicting the relationship between the percentage ofthe dose excreted in rat bile in vivo and in vivo intrinsic biliaryclearance and in vitro biliary clearance in 96-hr sandwich culturedhepatocytes for the following model substrates: inulin (□), salicylate(♦), methotrexate (◯), [D-pen^(2,5)]enkephalin (▴), and taurocholate(). The in vivo intrinsic biliary clearance was calculated fromEquation 2 based on in vivo biliary clearance values from theliterature. The in vitro biliary clearance was calculated from Equation4. The broken line is the fit of a linear regression equation to thedata;

FIG. 7A is a graph depicting cumulative uptake of [³H]264W94 (3 μM) instandard buffer (closed symbols) and Ca⁺⁺-free buffer (open symbols) inhepatocyte monolayers cultured for 3 hr;

FIG. 7B is a graph depicting cumulative uptake of [³H]264W94 (3 μM) instandard buffer (closed symbols) and Ca⁺⁺-free buffer (open symbols) inhepatocytes cultured in a sandwich configuration for 96 hr;

FIG. 8A is a graph depicting cumulative uptake of [³H]2169W94 (3 μM) instandard buffer (closed symbols) and Ca⁺⁺-free buffer (open symbols inhepatocyte monolayers cultured for 3 hr; and

FIG. 8B is a graph depicting cumulative uptake of [³H]2169W94 (3 μM) instandard buffer (closed symbols) and Ca⁺⁺-free buffer (open symbols) inhepatocytes cultured in a sandwich configuration for 96 hr;

FIG. 9A presents the chemical structures of the compound 264W94, whereinthe asterisk sign indicates the position of ¹⁴C incorporated uniformly;and

FIG. 9B presents the chemical structures of the compound 2169W94,wherein the asterisk sign indicates the position of ¹⁴C incorporateduniformly.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a method is provided for thescreening of a candidate compound or substrate for susceptibility tobiliary excretion. The method comprises the steps of providing a cultureof hepatocytes, the culture comprising at least one bile canaliculushaving a canalicular space; exposing a candidate compound to theculture; and determining an amount of the candidate compound in thecanalicular space of the at least one bile canaliculus, the amount ofthe candidate compound in the canalicular space of the at least one bilecanaliculus indicating the susceptibility of the candidate compound tobiliary excretion.

As would be appreciated by one of ordinary skill in the art, in vivobiliary excretion of substrates involves translocation across thesinusoidal membrane, movement through the cytoplasm, and transportacross the canalicular membrane. Thus, in a preferred hepatocyte cultureof the present invention, functional properties displayed by hepatocytesin vivo are established. For example, the establishment of hepatictransport systems, such as sinusoidal or canalicular transport systems,or both sinusoidal and canalicular transport systems is particularlycontemplated in accordance with the present invention. Exemplarytransport systems include, but are not limited to, Ntcp, cMOAT, OATP1,OATP2, MRP2, P-gp, BSEP and MDR2.

Additionally, the establishment of at least one bile canaliculus and theestablishment of normal hepatocyte morphology in the hepatocyte culturesare also contemplated in accordance with the present invention.Preferably, the culture comprises a plurality of bile canaliculi. Morepreferably, the plurality of bile canaliculi comprise a canalicularnetwork. The amount of candidate compound, as discussed in detail below,in the canalicular space of the at least one bile canaliculus indicatesthe susceptibility of the candidate compound to biliary excretion.

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the invention.

The term “candidate compound” or “candidate substrate” is meant to referto any compound wherein the characterization of the compound'ssusceptibility to biliary excretion is desirable. Exemplary candidatecompounds or substrates include xenobiotics such as drugs and othertherapeutic agents, carcinogens and environmental pollutants, as well asendobiotics such as steroids, fatty acids and prostaglandins.

The candidate drugs and other therapeutic agents screened in accordancewith the method of the present invention are contemplated to be usefulin the treatment of warm-blooded vertebrates. Therefore, the inventionconcerns mammals and birds.

Contemplated is the treatment of mammals such as humans, as well asthose mammals of importance due to being endangered (such as Siberiantigers), of economical importance (animals raised on farms forconsumption by humans) and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans (such ascats and dogs), swine (pigs, hogs, and wild boars), ruminants (such ascattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also contemplated is the treatment of birds, including thetreatment of those kinds of birds that are endangered, kept in zoos, aswell as fowl, and more particularly domesticated fowl, i.e., poultry,such as turkeys, chickens, ducks, geese, guinea fowl, and the like, asthey are also of economical importance to humans. Thus, contemplated isthe treatment of livestock, including, but not limited to, domesticatedswine (pigs and hogs), ruminants, horses, poultry, and the like.

The term “biliary excretion” is meant to refer to a biological processwherein substances are removed from an animal's circulatory system bybeing taken up by hepatocytes (liver cells) and excreted in bile via thebile canaliculi. Uptake into the hepatocytes is mediated by transportsystems endogenous to hepatocytes, including, but not limited to, Ntcpand OATP1. Bile canaliculi are structures within liver tissue whichreceive excreted components from the hepatocytes and transport the bileto a bile duct for removal from the animal.

By the phrase “an amount of candidate compound” and/or the phrase“determining an amount of candidate compound in the at least one bilecanaliculus”, as used herein and in the claims, it is meant to refer toany amount of candidate compound that is taken up by hepatocytes andexcreted into the at least one bile canaliculus in accordance with theassay of the present invention. For example, “an amount” can refer tosubstantially no candidate compound residing in the at least one bilecanaliculus after exposure of a candidate compound to a culture inaccordance with the present invention. Alternatively, “an amount” canrefer to substantially all of the candidate compound residing in the atleast one bile canaliculus after exposure of a candidate compound to aculture in accordance with the present invention. Thus, the phrase “anamount of candidate compound in the at least one bile canaliculus” canbe used to describe candidate compounds that are not highly excreted,extensively excreted, and extensively and rapidly excreted.

The phrase “determining an amount of candidate compound in the at leastone bile canaliculus” is also meant to refer to the use of a biliaryexcretion index calculation and a biliary clearance calculation asdescribed herein below. The phrase “determining an amount of a candidatecompound in the at least one bile canaliculus” may also refer to thedetection of a reduced amount of a marker compound due to uptake ofcandidate compound into the at least one bile canaliculus as describedin the high throughput embodiment of the assay of the present inventiondescribed herein below. Thus, quantitative and qualitativedeterminations of “an amount of candidate compound in the at least onebile canaliculus” are contemplated to be within the scope of the presentinvention.

The phrase “an amount of candidate compound” and/or the phrase“determining an amount of candidate compound in the at least one bilecanaliculus” are also meant to refer to the screening of, for example, aclass or series of candidate compounds and then establishing a rankingof susceptibility to biliary excretion of the candidate compounds withinthe class or series. It is thus contemplated in accordance with apreferred embodiment of the present invention that the candidatecompound or compounds wherein lesser or lower susceptibility toexcretion is observed according to such a ranking may be chosen forfurther experimentation or development as a therapeutic agent, whilecompounds wherein higher or greater susceptibility to excretion isobserved according to such a ranking may be excluded from furtherexperimentation or development as a therapeutic agent.

However, as would be readily apparent to one of ordinary skill in theart, the characteristic that a compound is susceptible to biliaryexcretion does not necessarily preclude further development of thecompound as a therapeutic agent. Indeed, the decision of whether toproceed with the development of a particular candidate compound as atherapeutic agent is based on many factors, including, but not limitedto, the biological activity of the candidate compound. Whilesusceptibility to biliary excretion is an important factor, it is notthe only factor that is typically considered by one of ordinary skill inthe art. Characterization of susceptibility to biliary excretion inaccordance with the method of the present invention thus provides datathat is desirable for use by one of ordinary skill in the art inevaluating whether to proceed with the development of a candidatecompound as a therapeutic agent.

The term “marker compound” is meant to refer to a chemical compound thatis readily detectable using a standard detection technique, such asfluorescence or chemiluminescence spectrophotometry, scintillationspectroscopy, chromatography, liquid chromatography/mass spectroscopy(LC/MS), colorimetry, and the like. Exemplary marker compounds thusinclude, but are not limited to, fluorogenic or fluorescent compounds,chemiluminescent compounds, calorimetric compounds, UV/VIS absorbingcompounds, radionuclides and combinations thereof.

Therapeutic compositions that are taken up and excreted extensivelythough the biliary excretion processes described herein typically have aminimal chance of imparting therapeutic effects in a subject. It is thusvery desirable to establish an in vitro test for a compound'ssusceptibility to hepatocyte uptake and biliary excretion so as tofacilitate elimination of a compound with an undesirably highsusceptibility from further evaluation as a therapeutic agent early inthe evaluation process. The biliary excretion assay of the presentinvention provides such a test.

Rat hepatocytes are preferred in a culture for use in the method of thepresent invention; but, any suitable source of hepatocytes as would beapparent to one of ordinary skill in the art is contemplated to bewithin the scope of the present invention. Exemplary sources include thewarm-blooded vertebrates listed above. In particular, exemplary sourcesinclude, but are not limited to, human beings, monkeys, apes, cats,dogs, pigs, hogs, cattle, oxen, sheep, horses, turkeys, chickens, ducksand geese.

The biliary excretion assay method of the present invention mayoptionally comprise establishing a sandwich culture of hepatocyteswherein at least one hepatocyte layer is formed between two layers ofmatrix. While configuration as a sandwich culture is the preferredconfiguration for the culture, any suitable configuration as would beapparent to one of ordinary skill in the art is contemplated to bewithin the scope of the present invention. For example, clusters,aggregates or other associations or groupings of hepatocytes in aculture wherein at least one bile canaliculus is formed and whereinfunctional properties of hepatocytes are established are contemplated tofall within the scope of the present invention. Preferably, the cultureconfiguration facilitates the formation of a plurality of bilecanaliculi. More preferably, the culture configuration facilitates theformation of a canalicular network. The amount of candidate compound, asdiscussed in detail herein, in the canalicular space of the bilecanaliculi indicates the susceptibility of the candidate compound tobiliary excretion.

Additionally, in the preferred sandwich configuration, hepatocytes arecultured in monolayers between two layers of matrix or scaffolding. But,the hepatocytes can also be embedded in the matrix or can extendnon-uniformly through the matrix vertically, horizontally, diagonally,or in any combination thereof, such that one-dimensional,two-dimensional and three-dimensional hepatocytes aggregates are formed.In accordance with the present invention, it is thus contemplated thatthe hepatocyte cultures can be formed by mixing hepatocyte cells with anappropriate matrix and inserting the mixture into a suitable culturecontainer, such as a multi-well plate.

While collagen is a preferred substrate or scaffolding for the cultureof hepatocytes, any suitable substrate or scaffolding whether natural,synthetic or combinations thereof as would be apparent to one ofordinary skill in the art is contemplated to be within the scope of thepresent invention. For example, other biological substrates, includingbut not limited to laminin and the basement membrane derived biologicalcell culture substrate sold under the registered trademark MATRIGEL® byCollaborative Biomedical Products, Inc. of Bedford, Mass., arecontemplated to comprise suitable substrate or scaffolding material.Synthetic matrix materials, substrate materials or scaffoldingmaterials, which are typically made from a variety of materials such aspolymers, are also contemplated to fall within the scope of the presentinvention. The variation of component materials with a particular matrixfor use in culturing hepatocytes is also contemplated in accordance withthe method of the present invention.

The cultured hepatocytes are preferably cultured as a “long-termculture”. By “long-term culture” it is meant to refer to hepatocytesthat have been cultured for at least about 12 hours. More preferably, by“long-term culture” it is meant to refer to hepatocytes that have beencultured for at least about 24 hours, for at least about 48 hours, orfor at least about 72 hours. Even more preferably, by “long-termculture” it is meant to refer to hepatocytes that have been cultured forat least about 96 hours. Long-term culturing facilitates the formationof bile canaliculi and the establishment of functional properties withinthe hepatocytes.

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

Side-by-Side Embodiment

In accordance with one embodiment of the present invention, replicatehepatocyte cultures are established, preferably in sandwichconfiguration. A first culture is exposed to a standard buffer and asecond culture is exposed to a Ca⁺⁺-free buffer. Exposure to theCa⁺⁺-free buffer disrupts the bile canaliculi within the hepatocytemonolayers by breaking down adhesional processes or junctional complexesin the monolayer of hepatocytes. While exposure to the Ca⁺⁺-free bufferis a preferred method of breaking down the adhesional processes orjunctional complexes to substantially disrupt the bile canaliculi, anysuitable technique for breaking down the adhesional processes orjunctional complexes to promote substantial disruption of the bilecanaliculi as would be apparent to one of ordinary skill in the art iscontemplated to be within the scope of the present invention. Exemplarytechniques include, but are not limited to, the administration to theculture of peptides which interact with cell-to-cell binding sites tothereby prevent neighboring cells from binding.

A candidate compound or compounds is/are then added to each culture. Thecandidate compound(s) cannot be retained within the bile canaliculi inthe culture that was treated with Ca⁺⁺-free buffer. Thus, in thisculture, candidate compound(s) may be taken up into the hepatocytes andretained within the cytoplasm of the hepatocytes. However, any amount ofthe candidate compound(s) that is excreted by the hepatocytes across thecanalicular membrane will flow into the buffer medium and will beremoved when the buffer medium is removed. In contrast, when candidatecompound(s) is/are administered to the hepatocyte sandwich culture inwhich the bile canaliculi are intact, any candidate compound(s) thatis/are taken up by the cells and excreted by the cells is/are maintainedboth in the cytoplasm of the hepatocytes and in the bile canaliculi.

It is then desirable to obtain a measurement of the amount of candidatecompound present within the intact bile canaliculi. The buffer media isremoved from the cultures and the cultures are washed and lysed. Asdescribed in the Laboratory Examples presented herein below, the lysingof the cells within the cultures may be accomplished by addition of asuitable lysis buffer coupled with agitation of the culture. A preferredlysis buffer includes a detergent. The desired measurement is obtainedby comparing the amount of candidate substance present in the lysatefrom the culture which has disrupted bile canaliculi (such as byexposure to Ca⁺⁺-free medium) as compared to the lysate from the culturewith intact bile canaliculi. Two particular calculations have beenutilized to compare the cultures and to determine an amount of thecandidate compound residing in the intact bile canaliculi. As describedabove, the amount of candidate compound in the intact bile canaliculiindicates the candidate compound's susceptibility to biliary excretion.

One calculation is described as a biliary excretion index, which is acalculation of the uptake and excretion of the candidate compound asfollows: 100%×{(uptake in the culture with intact bile canaliculi minusuptake within hepatocytes only in the Ca⁺⁺-free culture) divided by(uptake in the culture with intact bile canaliculi)}. The othercalculation is a biliary clearance calculation, which is performed asfollows: (uptake in the culture with intact bile canaliculi minus uptakewithin hepatocytes only in the Ca⁺⁺-free culture) divided by (time ofincubation multiplied by the concentration of the candidate compound inthe buffer medium).

Upon comparison of the in vitro assay of the present invention to astandard in vivo assay for biliary excretion as described in theLaboratory Examples presented herein below, it was determined thatbiliary clearance provided a more accurate and desirable evaluation ofexcretion. Particularly, the in vitro biliary clearance calculationadequately differentiated among candidate substances that are: (1) nothighly excreted; (2) extensively excreted; and (3) extensively andrapidly excreted. Thus, the use of the biliary clearance calculationcomprises an important aspect of the invention.

Metabolite Assay Embodiment

In the hepatocytes of the method of the present invention certainmetabolic activities (called Phase I activities) may be substantiallyreduced. The substantial reduction in metabolic activity coupled withmaintenance of biliary transport represents an advantage of the in vitrobiliary excretion assay of the present invention in that adifferentiation can be made between biliary excretion of a parentcandidate compound versus a metabolite or metabolites of the parentcandidate compound. This feature comprises an important aspect of thepresent invention.

In accordance with a preferred embodiment of the metabolite assay of thepresent invention, the method comprises establishing a first set andsecond set of two cultures of hepatocytes, with each culture preferablycomprising at least one layer of hepatocytes sandwiched between twolayers of collagen and at least one bile canaliculus formed within atleast one layer of hepatocytes. The first set of cultures includesintact bile canaliculi and the second set of cultures includes disruptedbile canaliculi.

Metabolic enzyme activity and/or transport systems are then induced inthe hepatocytes of one of the cultures within each of the first set andsecond set of cultures in accordance with art-recognized techniquesusing inducers which are known to up-regulate Phase I hepatic enzymeactivity, such as phenobarbital and β-naphthoflavone. Exemplary inducersand techniques associated with the same are described by Parkinson, A.(1996) Biotransformation of Xenobiotics in Casarett and Doull'sToxicology. The Basic Science of Poisons., 5^(th) Ed. (Klaassen, C. D.ed.) pp. 113-186, McGraw Hill, N.Y., and by LeCluyse et al., (1996)Cultured rat hepatocytes, in Models for Assessing Drug Absorption andMetabolism (Borchard et al. eds), pp 121-160, Plenum Press, New York,the contents of each of which are herein incorporated by reference.

A candidate parent compound is exposed to the first and second sets ofcultures for a time sufficient to allow uptake of the candidate parentcompound. Each set of cultures is washed and then lysed. The amount ofcandidate parent compound present in the lysate obtained from theculture in each set of cultures having inactive metabolic enzymes isdetermined. The amount of metabolite of the candidate parent compoundpresent in the lysate obtained from the culture in each set of cultureshaving active metabolic enzymes is also determined.

A biliary clearance value for the cultures having inactive metabolicenzymes is calculated using the amount of candidate parent compound inthe culture lysate. The calculated biliary clearance value is then usedto determine the susceptibility of the candidate parent compound tobiliary excretion, as described above. A biliary clearance value for thecultures having active metabolic enzymes is calculated using the amountof metabolite of the candidate parent compound in the culture lysate.The calculated biliary clearance value is then used to determine thesusceptibility of the metabolite to biliary excretion, as describedabove. This information is contemplated to be useful, for example, inevaluating whether or not to administer a therapeutic composition in apro-drug form.

High Throuqhput Assay Embodiment

An additional alternative embodiment of the present invention pertainsto a high throughput hepatic uptake and biliary excretion assay. Such anassay preferably involves the use of cultured hepatocytes as describedabove, in conjunction with a marker compound that is a substrate forendogenous sinusoidal or canalicular transport systems, or bothsinusoidal and canalicular transport systems. Exemplary transportsystems include, but are not limited to, Ntcp, cMOAT, OATP1, OATP2,MRP2, P-gp, BSEP and MDR2. Particularly, a candidate compound isadministered to a hepatocyte culture in conjunction with a markercompound in accordance with the cell culture and compound administrationtechniques described in the Laboratory Examples presented below.

Uptake and excretion competition between a candidate compound and themarker compound is then evaluated. That is, a significant drop in theamount of marker compound (e.g. measured or detected signal from themarker compound) within bile canaliculi in a culture may indicate thatthe candidate compound (as opposed to the marker compound) is taken upand excreted extensively.

A ranking of susceptibility to hepatic uptake and biliary excretion ofthe candidate compounds is then established. It is thus contemplated inaccordance with a preferred embodiment of the high throughput assay ofthe present invention that the candidate compound or compounds whereinlesser or lower susceptibility to excretion is observed according tosuch a ranking may be chosen for further experimentation or developmentas a therapeutic agent, while compounds wherein higher or greatersusceptibility to excretion is observed according to such a ranking maybe excluded from further experimentation or development as a therapeuticagent.

An exemplary marker compound comprises the fluorescent cMOAT/MRP2substrate, carboxydichlorofluorescein. Preferably,carboxydichloroflorescein diacetate, which exhibits only a weakfluorescence, is utilized as a fluorogenic precursor due to its rapidpenetration into the hepatocyte plasma membrane.Carboxydichlorofluorescein diacetate is hydrolyzed readily in thecytoplasm of hepatocytes by intracellular esterases to a highlyfluorescent product, carboxydichlorofluorescein as described inHaugland, Molecular Probes: Handbook of Fluorescent Probes and ResearchChemicals (1992-1994), p. 134, Molecular Probes, Inc., 1992.

The fluorescence of carboxydichlorofluorescein is sensitive to pH andthus any assay based on the intensity of carboxydichlorofluorescenceshould consider the effects of pH. However, it has been observed thatless than a 0.3 pH unit difference has been found between cytosol andbile canaliculi in hepatocyte couplets. Althoughcarboxydichlorofluorescein has been used for pH determinations in acidicorganelles, its fluorescence intensity is not altered markedly betweenpH 7.1 and pH 7.4. The fluorescence of carboxydichlorofluorescein at pH7.4 is only about 10-20% higher than at pH 7.1 at maximum emissionwavelength. Inasmuch as the fluorescence of carboxydichlorofluoresceinis used as a qualitative probe to localize carboxydichlorofluoresceincellular distribution, the slight pH gradient between cytosol and thecanaliculi do not affect the application of the high throughput assay ofthe present invention.

Additional marker compounds include, but are not limited to,fluorescein-labeled taurocholate, a bile acid that is rapidly andextensively taken up by hepatocytes and excreted into the bilecanaliculi as described in the Laboratory Examples presented hereinbelow; cholylglycylamido fluorescein, another fluorescent bile aciddescribed by Boyer and Soroka, Gastroenterology 109:1600-1611 (1995);rhodamine 123 for MDR2 and P-gp; and carboxyfluorescein diacetate(CFDA).

It is contemplated that the method of the present invention may beperformed within standard multi-well assay plates as are well known inthe art, such as the 96-well micro-titer plates that are available fromICN Pharmaceuticals, Inc. (Costa Mesa, Calif.). Thus, a plurality ofcandidate compounds can be simultaneously screened for susceptibility tobiliary excretion within multiple wells of a multi-well plate.

The following Laboratory Examples have been included to illustratepreferred modes of the invention. Certain aspects of the followingLaboratory Examples are described in terms of techniques and proceduresfound or contemplated by the present inventors to work well in thepractice of the invention. These Laboratory Examples are exemplifiedthrough the use of standard laboratory practices of the inventors. Inlight of the present disclosure and the general level of skill in theart, those of skill will appreciate that the following LaboratoryExamples are intended to be exemplary only and that numerous changes,modifications and alterations can be employed without departing from thespirit and scope of the invention.

Laboratory Examples

The following Laboratory Examples pertain to the establishment of acorrelation of biliary excretion in sandwich-cultured rat hepatocytes(present method) and in vivo in rats (standard). Five model substratesrepresenting a diverse spectrum of biliary excretion properties wereselected to examine the relationship between the percentage of the doseexcreted in bile in vivo in rats and in vitro using sandwich-culturedhepatocytes in accordance with the methods of the present invention. Thefive model substrates included inulin, salicylate, methotrexate,[D-pen^(2,5)]enkephalin and taurocholate.

Additionally, a comparison of in vivo and in vitro biliary excretion of264W94 and its metabolites is set forth in Example 4. Compound 2169W94is the O-demethylated metabolite of 264W94 in rats and humans, which canundergo further conjugation with urindine-5′-diphosphoflucuronic acid toform a glucuronide conjugate (Silveret al., ISSX Proceedings, (SanDiego, Calif. USA) pp. 387, 1996). The structural formulas of compounds264W94 and 2169W94 are presented in FIG. 9.

Materials and Methods Used in the Examples

Chemicals. [³H]Taurocholate (3.4 Ci/mmol; purity>97%), -[¹⁴C]salicylate(55.5 mCi/mmol; purity>99%), and [³H][D-pen^(2,5)]enkephalin (36Ci/mmol; purity>97%0 were obtained from Dupont New England Nuclear(Boston, Mass.). [³H]Methotrexate (13.7 Ci/mmol; purity>99%) and[³H]inulin (1.3 Ci/mmol; purity 97%) were obtained from AmershamInternational plc (Buckinghamshire, England). Compounds [¹⁴C]264W94((3R,5R)-3-butyl-3-ethyl-2,3,4,5-tetrahydro-7,8-dimethoxy-5-phenyl-1,4-benzothiazepine-1,1-dioxide;45.5 mCi/mmol; purity>99%) and [¹⁴C]2169W94 ((3R,5R)-3-ethyl-2,3,4,5-tetrahydro-7-methoxy-8-hydroxy-5-phenyl-1,4-benzothiazepine-1,1-dioxide;43.7 mCi/mmol; purity>99%) were obtained from Glaxo Wellcome, Inc.(Research Triangle Park, N.C.). Collagenase (type I, class I) wasobtained from Worthington Biochemical Corp. (Freehold, N.J.). Dulbecco'smodified Eagles's medium (DMEM), fetal bovine serum and insulin werepurchased from Gibco (Grand Island, N.Y.). Rat tail collagen (type I)was obtained from Collaborative Biomedical Research (Bedford, Mass.).All other chemicals and reagents were of analytical grade and werereadily available from commercial sources.

Animals. Male Wistar rats (250-280 g) from Charles River Laboratory(Raleigh, N.C.) were used as liver donors. Rats were housed individuallyin stainless-steel cages in a constant alternating 12-hr light and darkcycle at least 1 week before the study was performed, and were fed adlibitum until use. Bile duct cannulated rats (200-250 g) were obtainedfrom Charles River (Raleigh, N.C.). All procedures were approved by theInstitutional Animal Care and Use Committee at the University of NorthCarolina at Chapel Hill, Chapel Hill, N.C.

Preparation of Culture Dishes. Plastic culture dishes (60 mm) wereprecoated with rat tail collagen at least 1 day prior to preparing thehepatocyte cultures. To obtain a gelled collagen substratum, ice-coldneutralized collagen solution (0.1 ml, 1.5 mg/ml, pH 7.4) was spreadonto each culture dish. Freshly coated dishes were placed at 37° C. in ahumidified incubator for approximately 1 hr to allow the matrix materialto gel, followed by addition of 3 ml DMEM to each dish and storage in ahumidified incubator.

Culture of Rat Hepatocytes. Hepatocytes were isolated with a two-stepperfusion method. Briefly, rats were anesthetized with ketamine andxylazine (60 and 12 mg/kg i.p., respectively) prior to portal veincannulation. The liver was perfused in situ with oxygenated Ca²⁺-freeKrebs-Henseleit bicarbonate buffer containing collagenase type I (0.5mg/ml) for 10 min. The hepatic capsule was removed with forceps. Thehepatocytes were released by shaking the liver gently in 100 ml DMEM.

The released cells were filtered through a sterile nylon mesh (70-μm).The hepatocyte suspensions were centrifuged at 50× g for 3 min. The cellpellet was resuspended in 25 ml DMEM and an equal volume of 90% isotonicpolyvinylpyrrolidone-coated silica colloid centrifugation medium (pH7.4) sold under the registered trademark PERCOLL® by Pharmacia, Inc. ofPiscataway, N.J. The resulting cell suspension was centrifuged at about70 to about 150× g for 5 min. The pellet was resuspended in 50 ml DMEMand the cell suspensions were combined into one tube followed bycentrifugation at 50× g for 3 min. Hepatocyte viability was determinedby trypan blue exclusion. Only those hepatocyte preparations withviability greater than 90% were utilized for further studies.

Hepatocyte suspensions were prepared with DMEM containing 5% fetal calfserum, 1 μM dexamethasone and 4 mg/L insulin. Hepatocyte suspensionswere added to the precoated dishes at a density of about 2-3×10⁶cells/60-mm dish. Approximately 1 hr after plating the cells, the mediumwas aspirated and 3-ml fresh DMEM was added. For transport studies,hepatocytes that had been seeded for 3-5 hr without collagen overlaywere defined as 3-hr or short-term cultured hepatocytes.

To prepare sandwich-cultured hepatocytes, neutralized collagen solution(0.1 ml, about 1.5 to about 3.0 mg/ml, pH 7.4) was added to themonolayers 24 hr after the cells were seeded. Cultures with collagenoverlay were incubated for 45 min at 37° C. in a humidified incubator toallow the collagen to gel before addition of DMEM. Medium was changed ona daily basis until the fourth day after the cells were seeded. Thesehepatocytes were referred to as 96-hr or long-term cultured hepatocytes.

Cumulative Uptake Studies in Sandwich-Cultured Hepatocytes. Hepatocytescultured in a collagen-sandwich configuration were incubated in 3 mlstandard buffer or Ca²+-free buffer at 37° C. for 10 min. After removingthe incubation buffer, uptake was initiated by addition of 3 ml standardbuffer containing substrate to each dish. After incubation fordesignated times, cumulative uptake was terminated by aspirating theincubation solution and rinsing 4 times with 3 ml ice-cold standardbuffer to remove extracellular substrate. After washing, 2 ml of 1%Triton X-100 solution was added to culture dishes, and the cells werelysed by shaking the dish on a shaker for 20 min at room temperature. Analiquot (1 ml) of lysate was analyzed by liquid scintillationspectrometry. Bio-rad DC Protein Assay Kit (Bio-Rad Laboratories,Hercules, Calif.) was used to determine the protein concentration in theculture extracts using bovine serum albumin as standard. Triton X-100(1%) did not interfere with the assay. All values for substrate uptakeinto cell monolayers were corrected for nonspecific binding to thecollagen by subtracting the substrate uptake determined in theappropriate control dishes in the absence of cells as describedpreviously.

Biliary Excretion in Rats after Intravenous Administration of 264W94 andOral Administration of 2169W94. [¹⁴C]264W94 was formulated as a solutionin a mixture of sterile water/polypropylene glycol 400/ethanol (2:1:1v/v/v) at a concentration of 0.125 mg/mL. Following collection ofpre-dose bile, [¹⁴C]264W94 solution was administrated by caudal veininjection (0.1 mg/kg). For the 2169W94 studies, [¹⁴C]2169W94 wasprepared as a suspension at a concentration of 0.1 mg/mL in 0.5% (w/v)methylcellulose in water. Following collection of pre-dose bile,[¹⁴C]2169W94 suspension was administrated by gavage (1.0 mg/kg). Allrats were placed into individual plastic metabolism cages that allowedthe rats unrestrained movement. Bile was collected into polypropylenecontainers surrounded by ice. For the 264W94 studies, the bile containerwas changed at 8 and 24 hours after the dose. Previous studies indicatedthat samples were stable on ice for 24 hours. Bile samples were storedat −20° C. until analysis.

Analytical Procedure. Aliquots of cell lysate or bile samples containing264W94 or2169W94 were mixed with 2-fold volumes of ice-chilledacetonitrile, and centrifuged to remove precipitated proteins. Thesupernatant was evaporated under nitrogen at room temperature, andreconstituted in 100 μL of a 70/30 mixture of 50 mM ammoniumacetate/acetonitrile/trifluoroacetic acid (95:5:0.1 v:v:v) andacetonitrile. The sample extracts were injected onto a WATERS™ SYMMETRY™C18 column (3.9×150 mm) and eluted by a 85/15 mixture of 50 mM ammoniumacetate (pH 4.0) and acetonitrile; the percentage of acetonitrile wasincreased by a WATERS™ 600E System Controller to 55% over a period of 20minutes, and then to 100% during the next 10 minutes.

Radiocarbon that eluted from the HPLC was quantified with an on-lineradioactivity detector (RADIOMATIC FLO-ONE/BETA™ Radio-ChromatographyDetector Series 500 TR Series, Packard Instrument Co.). The peaks of264W94, 2169W94, and 2169W94 glucuronide were identified by comparingwith purified standard compound. Under these conditions, baselineseparation of these three components was achieved. The concentration ofthe three components was determined by normalizing the elutedradioactivity in each peak to the total injected radioactivity.

Data Analysis. Uptake data were normalized to the protein content andexpressed as mean ± SD from 3-4 separate preparations of hepatocytes.Statistical differences between mean values for the 10-min cumulativesubstrate uptake in the presence and absence of Ca²⁺ were determined bythe use of the well-known Student's t-test. A P value of <0.05 wasconsidered significant.

In vivo biliary clearance, Cl_(B) (ml/min/kg body weight), wascalculated according to Equation 1: $\begin{matrix}{{Cl}_{B} = \frac{{Amount}_{{bile}{({0 - T})}}}{{AUC}_{0 - T}}} & {{Equation}\quad 1}\end{matrix}$

where Amount_(bile(O-T)) represents the amount of parent drug recoveredin bile from 0 to time T when most drug was eliminated from the systemiccirculation, and AUC_(0-T) represents the area under the plasmaconcentration-time curve from 0 to time T (in minutes).

The in vivo intrinsic biliary clearance (Cl_(Bin), ml/min/kg bodyweight) was estimated according to Equation 2 based on the well-stirredmodel of hepatic disposition assuming biliary excretion is thepredominant elimination pathway (Pang et al., J. Pharmacokinet Biopharm.5:625-653, 1977). $\begin{matrix}{{Cl}_{Bin}\frac{Q \cdot {Cl}_{B}}{Q - {Cl}_{B}}} & {{Equation}\quad 2}\end{matrix}$

where Q represents rat hepatic plasma flow, 40 ml/min/kg of body weight{(blood flow×(1-hematocrit)}; Pollack et al., J. Pharmacol Exp. Ther.18:197-202, (1989), and Cl_(B) represents biliary clearance for modelcompounds reported in the literature or calculated from Equation 1.

Biliary excretion of substrates in the monolayers was quantitativelyassessed by the Biliary Excretion Index based on Equation 3:$\begin{matrix}{{BiliaryExcretionIndex} = {{\frac{{Uptake}_{standard} - {Uptake}_{{ca}^{++} - {free}}}{{Uptake}_{standard}} \cdot 100}\%}} & {{Equation}\quad 3}\end{matrix}$

where Uptake_(standard) and Uptake_(Ca) _(⁺⁺) _(-free) represent thecumulative uptake of substrate over a 10-min interval in the hepatocytemonolayers pre-incubated in standard buffer and in Ca⁺⁺-free buffer,respectively.

Biliary clearance in the sandwich-cultured hepatocytes, Cl_(B(culture))(ml/min/kg per body weight), was calculated according to Equation 4:$\begin{matrix}{{Cl}_{B{({culture})}} = \frac{{Uptake}_{standard} - {Uptake}_{{Ca}^{++} - {free}}}{{Time}_{incubation} \cdot {Concentration}_{medium}}} & {{Equation}\quad 4}\end{matrix}$

where Time_(incubation) was 10 min and Concentration_(medium)represented the initial substrate concentration in the incubationmedium. Rat liver weight and protein content in liver tissue wereassumed to be 40 g/kg of body weight and 0.20 g/g of liver weight(Seglen et al., Methods in Cell Biology (13^(th) Ed., Prescott D. M.Eds.) pp. 30-78, Academic Press, New York, 1976), respectively, in allcalculations.

Summary of the Results of the Examples

Biliary excretion of the five model substrates in long-termsandwich-cultured hepatocytes in accordance with the present inventionwas consistent with their in vivo biliary excretion properties.Quantification of biliary excretion in the cultured hepatocytesutilizing the biliary excretion index calculation is describedhereinabove. Briefly, the biliary excretion index represents thepercentage of retained substrate in the bile canaliculi. The results ofthe Laboratory Examples indicate that compounds undergoing negligiblebiliary excretion in vivo based on the percentage of dose excreted inbile (e.g., inulin, salicylate) have a low biliary excretion index(approximately zero). Compounds that are more extensively excreted inbile in vivo (e.g., methotrexate, [D-pen^(2,5)]enkephalin, andtaurocholate) have a high biliary excretion index (approximately 50%).

The relationship between the biliary excretion index and the percentageof the dose excreted in bile in vivo only reveals a categoricalcorrelation. Methotrexate and [D-pen^(2,5)]enkephalin representcompounds that are “highly” excreted in bile (approximately 60% and 70%of the i.v. dose was recovered in bile in 1 hr, respectively). Incontrast, taurocholate is “rapidly and extensively” excreted in thatalmost all of the i.v. dose was excreted in bile in less than 1 hr. Thebiliary excretion index can thus differentiate between compounds thatundergo extensive versus negligible or low biliary excretion.

However, the biliary excretion index appears unable to differentiatebetween compounds that are highly excreted in bile, like methotrexate(biliary excretion index: approximately 55%) or [D-pen^(2,5)]enkephalin(biliary excretion index: approximately 42%), and compounds that are“rapidly and extensively” excreted in bile, like taurocholate (biliaryexcretion index: approximately 56%). This limitation in the biliaryexcretion index may be due to the fact that this index is determinedpredominantly by the canalicular excretory functions. The percentage ofi.v.-administered substrate excreted into the bile in vivo isdertermined by sinusoidal uptake activity, canalicular excretoryactivity, as well as other competitive elimination processes.

Biliary clearance represents a more effective parameter for comparisonof the relationship between in vivo and in vitro biliary excretion. Thein vivo biliary clearance was calculated in the Laboratory Examples asthe ratio of the amount excreted into bile at time T and the plasma AUCbetween time 0 and time T. Because most of the administered dose waseliminated at time T, the biliary clearance approximates the biliaryclearance calculated from time 0 to time infinity. Biliary clearancecalculated in this matter is a function of intrinsic biliary clearanceand the hepatic plasma flow rate. To eliminate the effects of plasmaflow, the intrinsic biliary clearance was calculated based on the “wellstirred” model of hepatic disposition described by Pang and Rollan in J.Pharmacokinet. Biopharm. 5:625-653, 1977. Likewise, in vitro biliaryclearance was calculated as the ratio of the amount excreted in thecanalicular networks in the hepatocyte monolayers and the AUC in theincubation medium.

In the sandwich-cultured hepatocytes, the incubation medium wasaccessible to all hepatocytes in the dish at the same time. Thus, thecalculated in vitro biliary clearance should represent the intrinsicbiliary clearance. However, since biliary excretion involves twoprocesses, uptake across the sinusoidal membrane and excretion acrossthe canalicular membrane, the true intrinsic biliary clearance should bedetermined by transport across the canalicular membrane and calculatedbased on intracellular substrate concentrations. Therefore, the in vivoand in vitro “intrinsic” clearance values calculated in the LaboratoryExamples may be referred to as an “apparent” intrinsic biliary clearancevalue, which would be rate limited by the slowest step in the process,either sinusoidal uptake or canalicular excretion.

The correlation between in vitro biliary clearance and in vivo intrinsicbiliary clearance was high (Γ⁼0.9865) for the five model substrates.According to the in vivo intrinsic biliary clearance, the five modelsubstrates can be classified into three groups: compounds that are notexcreted in bile (inulin and salicylate; approximately 0 ml/min/kg),compounds that are highly excreted in bile (methotrexate and[D-pen^(2,5)]enkephalin, approximately 17.3 ml/min/kg and approximately34.4 ml/min/kg, respectively); and compounds that are rapidly andextensively excreted in bile (taurocholate, approximately 116.9ml/min/kg). The estimated in vitro biliary clearance adequatelydifferentiated between these three groups of compounds (approximately 0,4-13, and 56 ml/min/kg, respectively). These results suggest that thebiliary clearance more accurately characterizes the relationship betweenin vivo and in vitro biliary excretion as compared to the biliaryexcretion index.

EXAMPLE 1 Cumulative Uptake in Cultured Hepatocytes

The cumulative uptake of inulin was negligible (less than 0.01% ofinitial added substrate) at all incubation times in either short-term orlong-term cultured hepatocytes (FIGS. 1A and 1B). In the 3-hr culturedhepatocytes, the cumulative uptake of salicylate, methotrexate and[D-pen^(2.5)]enkephalin was not significantly different in standardbuffer and in Ca²⁺-free buffer (FIGS. 2A, 3A, and 4A; p>0.05). However,slightly higher cumulative uptake of taurocholate in standard buffercompared to Ca²⁺-free buffer was observed (FIG. 5A); at 10 min, thecumulative uptake in standard buffer was approximately 10% higher thanin Ca²⁺-free buffer (p=0.0352). In 96-hr cultured hepatocytes,extracellular Ca²⁺had no effect on the cumulative uptake of salicylate(FIG. 2B, p>0.05). However, the uptake of methotrexate,[D-pen^(2.5)]enkephalin, and taurocholate in long-term culturedhepatocytes in standard buffer was significantly higher than inCa²⁺-free buffer (FIG. 3B, 4B, and 5B; p<0.05).

EXAMPLE 2 Relationship Between the Percentage of Dose Excreted in Bilein Rats And Biliary Excretion Index in Cultured Hepatocytes

The five model substrates representing a diverse spectrum of biliaryexcretion properties were selected to examine the relationship betweenthe percentage of the dose excreted in bile in vivo in rats and theBiliary Excretion Index in sandwich-cultured hepatocytes. Informationregarding the percentage of the dose excreted in rat bile after i.v.administration was obtained from the literature. The extent of inulinand salicylate secretion into bile was negligible (Eriksson et al.,Acta. Physiol. Scand. 95:1-5, 1975; Laznicekand et al., Eur. J. DrugMet. Pharmacokinet. 19:21-26, 1994). Approximately 50-60% of a 22μmol/kg methotrexate dose (Bremnes et al., Cancer Res. 49:2460-2464,1989; Masuda et al., Cancer Res. 57:3506-10, 1997) and 70% of a 14.5μmol/kg [D-pen^(2,5)]enkephalin dose (Chen et al., Pharm. Res.14:345-350, 1997) were excreted into rat bile as unchanged drug in 1 hr.Taurocholate biliary excretion was more rapid and extensive thanmethotrexate and [D-pen^(2,5)]enkephalin. In 1 hr, virtually 100% of thedose (8.0 μmol/kg) was recovered in rat bile (Inoue et al., Biochim.Biophys. Acta. 833:211-216, 1985).

Biliary excretion in the sandwich-cultured hepatocytes can be expressedquantitatively as the Biliary Excretion Index calculated from Equation 3based on the 10-min cumulative uptake data in FIGS. 3B-5B. The BiliaryExcretion Index of inulin and salicylate was assumed to be negligiblebecause no statistically significant differences in the cumulativeuptake of inulin or salicylate were observed between standard buffer andCa²⁺-free buffer (p>0.05). The Biliary Excretion Index of methotrexate,[D-pen^(2,5)]enkephalin and taurocholate was 55.4±18.3%, 42.4±6.5% and56.4±5.2%, respectively. The relationship between the percentage of thedose excreted in rat bile in vivo and the Biliary Excretion Indexmeasured in the in vitro system is depicted in FIG. 6A. The BiliaryExcretion Index was very low for compounds undergoing negligible biliaryexcretion in vivo (e.g., inulin and salicylate). In contrast, theBiliary Excretion Index was moderately high for compounds that areexcreted in bile in vivo (e.g., methotrexate, [D-pen^(2,5)]enkephalin,and taurochloate).

EXAMPLE 3 Correlation of In Vitro and In Vivo Biliary Clearance

The in vivo biliary clearance (ml/min per kg body weight) of inulin,salicylate, methotrexate and taurocholate was 0.035 (Utesch et al.,Vitro Cell. Dev. Biol. 27A:858-863, 1991), ˜0 (Laznicekand et al., Eur.J. Drug Met. Pharmacokinet. 19:21-26, 1994), 12.1 (Masuda et al., CancerRes. 57:3506-10, 1997), and 29.8 (Inoue et al., Biochim. Biophys. Acta.833:211-216, 1985), respectively. In vivo biliary clearance of[D-pen^(2,5)]enkephalin, 18.5 ml/min/kg, was calculated based onEquation 1 from the data reported by Chen and Pollack (Chen and Pollack,Pharm. Res. 14:345-350, 1997). Based on these in vivo biliary clearancevalues, the intrinsic biliary clearance of inulin, salicylate,methotrexate, [D-pen^(2,5)]enkephalin and taurocholate was calculatedfrom Equation 2 (0.04, 0. 17.3, 34.4, and 116.9 ml/min/kg,respectively).

The in vitro biliary clearance of inulin, salicylate, methotrexate,[D-pen^(2,5)]enkephalin and taurocholate, calculated from Equation 4based on the 10-min cumulative uptake data (FIGS. 1B-5B) was ˜0, ˜0,4.1±1.0, 12.6±2.2, and 56.2±6.0 ml/min/kg, respectively. The in vivointrinsic biliary clearance correlated well with the in vitro biliaryclearance (Γ²=0.9865) for the five model compounds (FIG. 6B).

EXAMPLE 4 Comparison of In Vivo and In Vitro Biliary Excretion of 264W94and its Metabolites

The structural formulas of compounds 264W94 and 2169W94 are presented inFIG. 9. Compound 2169W94 is the O-demethylated metabolite of 264W94 inrats and humans, which can undergo further conjugation withurindine-5′-diphosphoflucuronic acid to form a glucuronide conjugate(Silver et al., ISSX Proceedings, (San Diego, Calif. USA) pp. 387,1996).

After i.v. administration of [¹⁴C]264W94 to rats (0.24 μmol/kg), neither264W94 nor 2169W94 was detected in bile in 24 hr. However, 35.4% (n=2)of the total administered radioactivity was recovered in bile in thefirst hour. Approximately, 30.0% of the radioactivity recovered in bilewas the 2169W94 glucuronide; the remaining 70% of radioactivity in bilerepresented unidentified metabolites. After oral administration of[¹⁴C]264W94 to rats (2.4 μmol/kg), 2169W94 was not detected in the bilein 24 hr. However, 66.4% (n=2) of the total administered radioactivitywas recovered in bile in 8 hr. Approximately, 88.7% of the radioactivityin bile was in the form of the 2169W94 glucuronide conjugate. These invivo results demonstrate that 264W94 and its O-demethylated product,2169W94, undergo negligible biliary excretion, but the glucouronideconjugate of 2169W94 undergoes extensive biliary excretion in rats.

To determine the biliary excretion of 264W94 and metabolites in 3-hr and96-hr cultured hepatocytes, hepatocyte monolayers were incubated instandard or Ca²⁺-free buffer before cumulative uptake was conducted instandard buffer containing 3 μM of [¹⁴C]264W94 or [¹⁴C]2169W94 (FIG. 7and 8). In 3-hr cultured hepatocytes, the cumulative uptake measured bytotal radioactivity of 264W94 or 2169W94 was similar in the hepatocytespre-incubated in standard buffer or Ca²⁺-free buffer (p>0.05),suggesting that the uptake of 264W94 and 2169W94 in short-term culturedhepatocytes was not affected by pre-incubation of the monolayers inCa²+-free buffer. In 96-hr cultured hepatocytes, the 1 0-min cumulativeuptake of 264W94 measured by total radioactivity was not significantlydifferent in the monolayers pre-incubated in standard buffer orCa²⁺-free buffer (p>0.05).

HPLC analysis of the cell lysate at 10 min revealed that 73.0% of thetotal radioactivity was in the form of 264W94 and 3.3% was the 2169W94glucuronide conjugate; 2169W94 was not detected in the lysate. In 96-hrsandwich-cultured hepatocytes, 10-min cumulative uptake of 2169W94 wasapproximately 70% greater in the presence of Ca²⁺ than in the absence ofCa²⁺ (p>0.05). In the 10-min cell lysate, approximately 16.7% of totalradioactivity was in the form of 2169W94, and approximately 58.5 was the2169W94 glucuronide conjugate. Compound 2169W94 forms the glucuronideconjugate which is excreted into bile canalicular networks in long-termcultured hepatocytes.

To further characterize the utility of the in vitro biliary excretionassay of the present invention to predict in vivo biliary excretion ofdrug metabolites, the in vitro and in vivo biliary excretion of 264W94,and its O-demethylated metabolites 269W694 and 2169W94 glucuronide wereexamined. Previous in vitro studies conducted with rat and human livermicrosomes, precision cut liver slices, and cDNA expressed hepaticcytochrome p450 isozymes indicated that 264W94 formed an O-demethylatedmetabolite at the 8-methoxy position. Among the several cytochrome p450isozymes examined, CYP3A4 was the isozyme primarily involved in themetabolism of 264W94 (Silver et al., ISSX Proceedings (San Diego, Calif.USA) p. 387, 1996).

In vivo disposition studies demonstrated that neither 264W94 nor itsO-demethylated metabolite, 2169W94, was excreted in the bile. But, the2169W94 glucuronide conjugate, along with other unidentifiedmetabolites, were extensively excreted in bile. The lack of biliaryexcretion of 264W94 in long-term sandwich-cultured hepatocytes wasconsistent with negligible in vivo biliary excretion of 264W94.

In vivo, approximately 35% of 264W94 equivalent was excreted in bile asmetabolites in 1 hr after i.v. administration of 264W94. In culturedhepatocytes, however, the biliary excretion of 264W94 metabolites wasnegligible (FIG. 7B). This apparent discrepancy between the in vivo andin vitro biliary excretion for metabolites of 264W94 may be explained bydifferences in metabolic activities. In vivo, 264W94 undergoesO-demethylation to form 2169W94; and subsequently, 2169W94 is conjugatedwith uridine-5′-diphosphoglucuronic acid to form 2169W94 glucuronide.This glucuronide conjugate accounts for 30% of the total amount excretedin bile. In the lysate of long-term sandwich-cultured hepatocytesincubated with 264W94, only approximately 3% of the total amountincubated was detected as the 2169W94 glucuronide conjugate. Theseresults indicated that the long-term cultured hepatocytes were notcapable of the O-demethylation reaction. Consequently, negligibleglucuronide conjugate was formed and excreted in the bile.

However, after incubation of the monolayers with 2169W94, theO-demethylated metabolite of 264W94, 58.5% of 2169W94 was converted toglucuronide conjugates and significant biliary excretion was observed inthe cultured hepatocytes (FIG. 8B). Evidently, phase I metabolicactivities such as O-demethylation deteriorate significantly, while thephase II metabolic activities such as glucuronide conjugation aremaintained, at least in part, in the long-term sandwich-culturedhepatocytes used in accordance with the present invention. Thus, thisLaboratory Example further indicates that the assay of the presentinvention can be employed to predict in vivo biliary excretion of asubstrate in its parent form. Indeed, the application of the present invitro assay method to study and to predict in vivo biliary excretion ofmetabolites requires consideration of the status of metabolic activitiesin the monolayers.

References

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

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It will be understood that various details of the invention may bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation—the invention being defined by theclaims.

What is claimed is:
 1. A method of screening a xenobiotic forsusceptibility to biliary excretion, the method comprising the steps of:(a) establishing first and second cultures of hepatocytes, each culturecomprising at least one bile canaliculus, the first culture havingintact bile canaliculi and the second culture having disrupted bilecanailculi; (b) exposing a xenobiotic to the first culture and to thesecond culture for a time (T) sufficient to allow uptake of thexenobiotic; (c) washing and then lysing the first and second cultures;(d) measuring an amount of xenobiotic present in a lysate obtained fromeach culture in step (c); (e) calculating a mass in the bile canaliculias the difference in the amount of xenobiotic present in the lysatesfrom the first culture having intact bile canaliculi and the secondculture having disrupted bile canaliculi; and (f) calculating a biliaryclearance value as the ratio of the mass in the bile canaliculi in step(e) and the area under the curve (AUC) in culture medium, wherein theAUC represents the inteqral of xenobiotic concentration in the mediumfrom time 0 to time T, to thereby screen the xenobiotic forsusceptibility to biliary excretion.
 2. The method of claim 1, whereinthe hepatocytes are isolated from a source selected from the groupconsisting of rat, human, monkey, ape, cat, dog, pig, hog, cattle, oxen,sheep, horses, turkeys, chickens, ducks and geese.
 3. The method ofclaim 1, wherein the first and second cultures of hepatocytes eachfurther comprise a long-term culture of hepatocytes.
 4. The method ofclaim 1, wherein the first and second cultures of hepatocytes furthercomprise a canalicular network.
 5. The method of claim 1, wherein thefirst and second cultures of hepatocytes are further characterized ashaving a configuration selected from the group consisting of clusters ofhepatocytes, aggregates of hepatocytes, at least one layer athepatocytes, and combinations thereof.
 6. The method of claim 1, whereinthe hepatocytes are embedded in a matrix.
 7. The method of claim 1,wherein the first and second cultures of hepatocytes further comprise asandwich culture of hepatocytes, the sandwich culture comprising atleast one layer of hepatocytes and at least one bile canaliculus withthe at least one layer of hepatocytes.
 8. The method of claim 7, whereinthe at least one layer of hepatocytes is sandwiched between two layersof matrix.
 9. The method of claim 7, wherein the first and secondcultures of hepatocytes each further comprise a long-term culture ofhepatocytes.
 10. The method of claim 6, wherein the matrix is selectedfrom the group consisting of a biological matrix medium, a syntheticmatrix medium, and combinations thereof.
 11. The method of claim 10,wherein the biological matrix medium is selected from the groupconsisting of collagens, laminins, basement membrane-derived complexes,derivatives thereof and combinations thereof.
 12. The method of claim 1,wherein steps (a) through (d) are carried out in at least one well of amulti-well plate.
 13. The method of claim 1, further comprisingscreening a plurality of xenobiotics simultaneously for susceptibilityto biliary excretion.
 14. The method of claim 1, further comprising thestep of differentiating between a xenobiotic that is not excreted inbile, a xenobiotic that is highly excreted in bile, and a xenobioticthat is readily and extensively excreted in bile.